Project Summary Regulation of the cell cycle of adult stem cells is crucial to maintaining tissue integrity. Cells irreversibly commit to division at the G1/S transition; however, how G1/S transition regulates adult stem cell cycles is currently unknown. I propose to study the molecular basis of how the G1/S transition is regulated in intestinal stem cell (ISC), which have physiologically high rates of proliferation. I will use intestinal organoid cultures as ex vivo models of ISC proliferation and intestinal homeostasis. Organoids are especially suited to this question because they are amenable to long-term live-cell imaging, while still retaining much of the stem cell physiology of their in vivo counterparts. My preliminary studies suggest that ISCs are especially sensitive to inhibition of Cyclin D/CDK4/6, a group of cyclin/CDK complexes that are key regulators of the G1/S transition. I propose to use live three-dimensional imaging intestinal organoids to investigate how the G1/S transition regulates ISC cell cycle and organoid homeostasis. (Aim 1) In collaboration with Dr. Prisca Liberali in the Friedrich Miescher Institute in Basel, I will learn and use light-sheet microscopy to perform long-term live-cell imaging on organoids expressing cell cycle and cell stemness reporters. This allows me to directly measure cell cycling dynamics in ICSs during CDK4/6 inhibition. For this aim, I will also develop computational image analysis tools to analyze large-scale 4-dimensional image data. (Aim 2) I will investigate the molecular mechanism that underlies G1/S transition in ISCs. To test that the cell cycle phenotype seen in ISCs during CDK4/6 inhibition is mediated by its known substrate, retinoblastoma protein (RB), I will specifically interfere with a motif in RB bound by Cyclin D, to see if RB phosphorylation specifically by Cyclin D/CDK4/6 is necessary for proper ISC G1/S control. Furthermore, I will test if expression levels of Cyclin D and RB also couple the G1/S transition to cell size in ISCs, by perturbing the expression levels of CDK4/6, Cyclin D, and RB, I and examining how ISC cell size distribution changes. Finally, I will investigate whether downstream cell fates are affected by ISCs that experience cell cycle disruption due to CDK4/6 inhibition, to see how cell-type homeostasis may also be affected by the G1/S transition. This project will elucidate the mechanisms used by adult stem cells to regulate their G1/S transition, an important problem in both developmental biology and cancer.